The palmityl binding sites of fatty acid synthetase from yeast.

Fatty acid synthetase was covalently labelled with [14C]palmitic acid from [14C]palmityl-CoA. Tryptic and peptic digestion of the [14C]palmityl enzyme resulted in the formation of radioactive palmityl peptides carrying the long-chain acyl residue both in oxygen-ester and thio-ester linkage. The lipophilic palmityl peptides were purified by column and thin-layer chromatography using organic lolvent systems. Peptides arising from the acyl carrier protein, the condensing enzyme and the palmityl transferase were identified and characterized. The amino acid sequence of a 4'-phosphopant-etheine-containing peptide was established. It comprises 13 residues and shows a high degree of homology with the acyl carrier protein from Escherichia coli. A heptapeptide and an octapeptide from the palmityl transferase active site were partially sequenced. The identical amino acid composition of palmityl transferase and malonyl transferase core peptides is briefly discussed.     

Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site

Pertussis toxin catalyzes the transfer of ADP-ribose from NAD to the guanine nucleotide-binding regulatory proteins Gi, Go, and transducin. Based on a partial amino acid sequence for a tryptic peptide of ADP-ribosylated transducin, asparagine had been characterized as the site of pertussis toxin-catalyzed ADP-ribosylation. Subsequently, cDNA data for the alpha subunit of transducin indicated that the putative asparagine residue was, in fact, not present in the protein. To determine the amino acid that served as the ADP-ribose acceptor, radiolabel from [adenine-U-14C]NAD was incorporated, in the presence of pertussis toxin, into the alpha subunit of transducin (0.3 mol/mol). An ADP-ribosylated, tryptic peptide was purified and fully sequenced by automated Edman degradation. The amino acid sequence, Glu-Asn 343-Leu-Lys-Asp 346-X-Gly 348-Leu-Phe, corresponds to the cDNA sequence coding the carboxyl-terminal nonapeptide, Glu 342-Phe 350, which includes by cDNA sequence cysteine at position 347. Neither Asn 343 nor Asp 346 appeared to be modified; residue 347 adhered to the sequencing resin. Cysteine, the missing residue, was eluted from the sequencing resin with acetic acid along with 76% of the peptide-associated radioactivity, half of which, presumably ADP-ribosylcysteine, eluted from an anion exchange column between NAD and ADP-ribose; the other half had a retention time corresponding to 5'-AMP. We conclude that Cys 347 and not Asn 343 or Asp 346 is the site of pertusis toxin-catalyzed ADP-ribosylation in transducin.     

Divergence in primary structure between the molecular forms of acetylcholinesterase

We have isolated a COOH-terminal tryptic peptide from the hydrophobic globular (5.6 S) form of Torpedo californica acetylcholinesterase that exhibits divergence in amino acid sequence from the catalytic subunit of the dimensionally asymmetric (17 S + 13 S) enzyme. The divergent peptide could be recovered from the glycophospholipid-modified 5.6 S enzyme only after treatment with phosphatidylinositol-specific phospholipase C. Upon reduction, carboxymethylation with [14C]iodoacetate, and trypsin digestion the resultant peptides were purified by gel filtration followed by high performance liquid chromatography. The high performance liquid chromatography profiles of 14C-labeled cysteine peptides from lipase-treated 5.6 S enzyme revealed unique radioactive peaks which had not been present in digests of the asymmetric form. These peaks all yielded identical amino acid sequences. The difference in chromatographic behavior of the individual peptides most likely reflects heterogeneity in post-translational processing. Gas-phase sequencing and composition analysis are consistent with the sequence: Leu-Leu-Asn-Ala-Thr-Ala-Cys. Composition includes 2-3 mol each of glucosamine and ethanolamine which is indicative of modification by glycophospholipid. Glucosamine is also present in an asparagine-linked oligosaccharide. The two forms of acetylcholinesterase diverge after the threonine residue within this peptide sequence; the hydrophobic form terminates with cysteine whereas the asymmetric form extends for 40 residues beyond the divergence. The locus of divergence and absence of any other amino acid sequence difference suggest that the molecular forms of acetylcholinesterase arise from a single gene by alternative mRNA processing.     

Sequence of active site peptides from the penicillin-sensitive D-alanine carboxypeptidase of Bacillus subtilis. Mechanism of penicillin action and sequence homology to beta-lactamases

It has been proposed that penicillin and other beta-lactam antibiotics are substrate analogs which inactivate certain essential enzymes of bacterial cell wall biosynthesis by acylating a catalytic site amino acid residue (Tipper, D.J., and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141). A key prediction of this hypothesis, that the penicilloyl moiety and an acyl moiety derived from substrate both bind to the same active site residue, has been examined. D-Alanine carboxypeptidase, a penicillin-sensitive membrane enzyme, was purified from Bacillus subtilis and labeled covalently at the antibiotic binding site with [14C]penicillin G or with the cephalosporin [14C]cefoxitin. Alternatively, an acyl moiety derived from the depsipeptide substrate [14C]diacetyl L-Lys-D-Ala-D-lactate was trapped at the catalytic site in near-stoichiometric amounts by rapid denaturation of an acyl-enzyme intermediate. Radiolabeled peptides were purified from a pepsin digest of each of the 14C-labeled D-alanine carboxypeptidases and their amino acid sequences determined. Antibiotic- and substrate-labeled peptic peptides had the same sequence: Tyr-Ser-Lys-Asn-Ala-Asp-Lys-Arg-Leu-Pro-Ile-Ala-Ser-Met. Acyl moieties derived from antibiotic and from substrate were shown to be bound covalently in ester linkage to the identical amino acid residue, a serine at the penultimate position of the peptic peptide. These studies establish that beta-lactam antibiotics are indeed active site-directed acylating agents. Additional amino acid sequence data were obtained by isolating and sequencing [14C]penicilloyl peptides after digestion of [14C]penicilloyl D-alanine carboxypeptidase with either trypsin or cyanogen bromide and by NH2-terminal sequencing of the uncleaved protein. The sequence of the NH2-terminal 64 amino acids was thus determined and the active site serine then identified as residue 36. A computer search for homologous proteins indicated significant sequence homology between the active site of D-alanine carboxypeptidase and the NH2-terminal portion of beta-lactamases. Maximum homology was obtained when the active site serine of D-alanine carboxypeptidase was aligned correctly with a serine likely to be involved in beta-lactamase catalysis. These findings provide strong evidence that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are related evolutionarily.     

Human placental estradiol 17 beta-dehydrogenase. Identification of a single histidine residue affinity-labeled by both 3-bromoacetoxyestrone and 12 beta-bromoacetoxy-4-estrene-3,17-dione

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity-labeled at pH 6.3 by 3-bromo[2'-14C]acetoxyestrone and 12 beta-bromo-[2'-14C] acetoxy-4-estrene-3,17-dione (both are substrates) in separate incubations. The affinity-alkylated enzyme samples were then treated separately as described below. Amino acid compositions of both samples revealed radioactive 3-carboxymethylhistidine. Tryptic digests of each sample were prepared, applied to Sephadex G-50, and 3-carboxymethylhistidine-bearing fractions identified. These peptides were further purified by cation exchange chromatography, gel filtration, and paper electrophoresis. The purified, 3-carboxymethylhistidine-bearing peptides labeled by the two steroids had identical electrophoretic mobilities at pH 6.5, 3.5, and 1.9. The amino acid sequence of the radioactive peptide alkylated by 3-bromo[2'-14C]acetoxyesterone was determined as: Leu-Ala-3-[14C]CmHis-Ser-Lys. The smaller quantity of peptide obtained from the inactivation with 12 beta-bromo[2'-14C]acetoxy-4-estrene-3,17-dione precluded the determination of its complete sequence. However, the first 3 residues were found to be Leu-Ala-3-[14C]CmHis and the amino acid composition showed that serine and lysine were also present. It is concluded that the steroid-binding site of human placental estradiol 17 beta-dehydrogenase contains a histidine residue which proximates the upper A-ring region of the steroid as it undergoes the reversible binding step.     

Cytochrome c-specific protein methylase III from Neurospora crassa.

A protein methylase III responsible for specifically methylating the cytochrome c in Neurospora crassa was partially characterized by using unmethylated horse heart cytochrome c as a substrate. This enzyme utilizes S-adenosyl-L-methionine as the methyl donor. An analysis of the distribution of [14C]methyl groups in the peptides obtained by chymotrypsin digestion of the enzymically methylated cytochrome c showed that all of the radioactivity could be recovered within a single peak after chromatography. This indicates that the enzyme methylates a specific amino acid sequence within cytochrome c. On hydrolysis of the radioactive chymotryptic peptide, Me-14C-labelled epsilon -N-mono-methyl-lysine, epsilon-N-dimethyl-lysine and epsilon-N-trimethyl-lysine were identified. The enzyme can easily be extracted from the N. crassa mycelial pads and was purified approx. 30-fold.     

Amino acid sequence at the ATP-binding site of cGMP-dependent protein kinase

The amino acid sequence at the ATP-binding site on the cGMP-dependent protein kinase has been determined. For this determination the enzyme was labeled covalently by 5'-p-fluorosulfonyl[14C]benzoyladenosine and fragmented using cyanogen bromide or digested by trypsin after succinylation. The 14C-labeled peptides were purified by gel filtration and high performance liquid chromatography. The amino acid sequence around the site was found to be: -Val-Glu-Leu-Val-Gln-Leu-Lys-Ser-Glu-Glu-Ser-Lys-Thr-Phe-Ala-Met-*Lys-Ile-Leu-Lys--Lys-Arg-His-Ile-Val-Asp-Thr-Arg-Gln-Gln-Glu-His-Ile-Arg-Ser-Glu-Lys-, in which *Lys is the lysine residue that was modified by the affinity reagent. When this sequence was compared with that of the ATP-binding site of the catalytic subunit of cAMP-dependent protein kinase, a high degree of structural homology was observed for this site in the two proteins.     

Isolation and identification of cysteinyl peptide labeled by 6- [( 4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate in the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase

6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate (6-BDB-TADP) has been shown to react at the reduced diphosphopyridine nucleotide (DPNH) inhibitory site of bovine liver glutamate dehydrogenase with incorporation of 1 mol of reagent/mol of enzyme subunit [Batra, S. P., & Colman, R. F. (1984) Biochemistry 23, 4940-4946]. The modified enzyme had lost one of the six free sulfhydryl groups per enzyme subunit as detected by 5,5'-dithiobis(2-nitrobenzoate). In the unmodified enzyme digested with trypsin, six cysteinyl peptides labeled with [14C]iodoacetic acid were detected by high-performance liquid chromatography (HPLC), whereas only five were observed in the 6-BDB-TADP-modified enzyme. A cysteinyl peptide has been isolated from modified enzyme digested with trypsin and chymotrypsin. Purification of the nucleotidyl peptide was accomplished by chromatography on phenyl boronate-agarose, followed by gel filtration on Sephadex G-25 and Bio-Gel P-4 in 50 mM ammonium bicarbonate, pH 8.0. The modified peptides were finally purified by HPLC on a C18 column using 0.1% trifluoroacetic acid with an acetonitrile gradient. By comparison of the amino acid composition and N-terminal residue of the isolated peptide with the known amino acid sequence of the enzyme, the peptide in the DPNH inhibitory site labeled by 6-BDB-TADP has been identified as the 19-membered fragment from Glu-311 to Lys-329. A unique residue, Cys-319, was identified as the reactive amino acid within the DPNH inhibitory site.     

Active site labeling of dopamine beta-hydroxylase by two mechanism-based inhibitors: 6-hydroxybenzofuran and phenylhydrazine

6-Hydroxybenzofuran and phenylhydrazine are mechanism-based inhibitors of dopamine beta-hydroxylase (D beta H; EC 1.14.17.1). We report here the isolation and characterization of radiolabeled peptides obtained after inactivation of D beta H with [3H]6-hydroxybenzofuran and [14C]phenylhydrazine followed by digestion with Staphylococcus aureus V8 protease. Inactivation of D beta H with [3H]6-hydroxybenzofuran gave only one labeled peptide, whereas inactivation with [14C]phenylhydrazine gave several labeled peptides. Each inhibitor labeled a unique tyrosine in the enzyme corresponding to Tyr477 in the primary sequence of the bovine enzyme (Robertson, J. G., Desai, P. R., Kumar, A., Farrington, G. K., Fitzpatrick, P. F., and Villafranca, J. J. (1990) J. Biol. Chem. 265, 1029-1035). In addition, [14C]phenylhydrazine also labeled a unique histidine (His249) as well as several other peptides. Examination of the complete peptide profile obtained by high pressure liquid chromatography analysis also revealed the presence of a modified but nonradioactive peptide. This peptide was isolated and sequenced and was identical whether the enzyme was inactivated by 6-hydroxybenzofuran or phenylhydrazine. An arginine at position 503 was missing from the sequence cycle performed by Edman degradation of the modified peptide, but arginine was present in the identical peptide isolated from native dopamine beta-hydroxylase. These data are analyzed based on an inactivation mechanism involving formation of enzyme bound radicals (Fitzpatrick, P. F., and Villafranca, J. J. (1986) J. Biol. Chem. 261, 4510-4518) interacting with active site amino acids that may have a role in substrate binding and binding of the copper ions at the active site.     

Affinity labeling of 3 alpha-hydroxysteroid dehydrogenase with 3 alpha-bromoacetoxyandrosterone and 11 alpha-bromoacetoxyprogesterone. Isolation and sequence of active site peptides containing reactive cysteines; sequence confirmation using nucleotide sequence from a cDNA clone

Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) of rat liver cytosol is potently inhibited at its active site by nonsteroidal anti-inflammatory drugs (NSAIDs). Using 3 alpha-bromoacetoxy-5 alpha-androstan-17-one (BrAnd, a substrate analog) and 11 alpha-bromoacetoxyprogesterone (Br11P, a glucocorticoid analog) as affinity-labeling agents, kinetic evidence was obtained that these agents alkylate this site. Inactivation of 3 alpha-HSD with either [14C]BrAnd or [14C] Br11P led to the incorporation of 1 mol of affinity-labeling agent per enzyme monomer. Complete acid hydrolysis of 3 alpha-HSD radiolabeled with either agent followed by amino acid analysis led to the identification of [14C]carboxymethylcysteine indicating that [14C]BrAnd and [14C]Br11P covalently tag discrete reactive cysteine(s) at the enzyme active site. Trypsin digestion of [14C]BrAnd-inactivated 3 alpha-HSD followed by peptide mapping led to the purification of a single radiolabeled peptide (3A1) which gave the following sequence: H2N-Ser-Ile-Gly-Val-Ser-Asn-Phe-Asn-X-Arg-CO2H. Identical experiments on [14C] Br11P-inactivated 3 alpha-HSD led to the purification of three radiolabeled peptides (11P1-11P3). The major radiolabeled peptide (11P1) had an identical sequence to 3A1 which was tagged with [14C]BrAnd. The minor radiolabeled peptides had the following sequences: H2N-Ser-Lys-Asp-Ile-Ile-Leu-Val-Ser-Tyr-X-Thr-Leu-Gly-Ser-Ser-Arg-CO2H (11P2) and H2N-Ser-Pro-Val-Leu-Leu-Asp-Asp-Pro-Val-Leu-X-Ala-Ile-Ala-Lys-CO2H (11P3). In each peptide group X was identified as carboxymethylcysteine. Alignment of the peptide sequences with the primary structure of 3 alpha-HSD, deduced from its cDNA clone, assigned peptide 11P1 to residues 162-171, peptide 11P2 to residues 208-223, and peptide 11P3 to residues 232-246 of the amino acid sequence. The reactive cysteines correspond to Cys170, Cys217, and Cys242. We propose that Cys170 labeled by BrAnd may lie within the catalytic pocket of the enzyme. By contrast the 11 alpha-bromoacetoxy group in Br11P labeled several reactive cysteines which may be involved in the binding of glucocorticoids and NSAIDs.     

Specific modification of the condensation domain of fatty acid synthase and the determination of the primary structure of the modified active site peptides

Fatty acid synthase from the uropygial gland of goose was inactivated by iodoacetamide with a second-order rate constant of 1.3 M-1 S-1 at pH 6.0 and 25 degrees C. Of the seven component activities of the synthase, only the condensation activity was significantly inhibited by iodoacetamide modification. Since preincubation of the enzyme with acetyl-CoA, but not with malonyl-CoA, protected the enzyme from inactivation by iodoacetamide, it is suggested that iodoacetamide probably modified the primer-binding thiol group at the condensation active site. Determination of the stoichiometry of modification was done using [1-14C]iodoacetamide that was purified by high-performance liquid chromatography. Graphical analysis of the data showed that binding of 1.2 carboxamidomethyl groups per subunit of fatty acid synthase would result in complete inhibition of the enzyme activity, suggesting that there is one condensation domain per subunit of fatty acid synthase. Analysis of the tryptic peptide map of the enzyme that was modified with [1-14C]iodoacetamide in the presence and absence of acetyl-CoA revealed that acetyl-CoA prevented the labeling of a major radioactive peptide and a minor radioactive peptide. These two peptides were purified by high-performance liquid chromatography. Amino acid analysis of these two peptides revealed that the major radioactive peptide contained S-carboxymethylcysteine while the minor radioactive peptide did not. However, the latter peptide contained beta-alanine, suggesting that this peptide was from the acyl carrier protein segment of fatty acid synthase and that the iodoacetamide treatment resulted in modification of the pantetheine thiol, although to a lower extent than the primer-binding thiol. The sequence of the primer-binding active site peptide from the condensation domain was H2N-Gly-Pro-Ser-Leu-Ser-Ile-Asp- Thr-Ala-Cys(carboxamidomethyl)-X-Ser-Ser-Leu-Met-Ala-Leu-Glu-Asn-A la-Tyr-Lys- COOH, the first reported sequence of the condensation active site from a vertebrate fatty acid synthase. The acyl carrier protein segment showed extensive sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the phosphopantetheine attachment, and the sequence was H2N-Asp-Val-Ser-Ser-Leu- Asn-Ala-Asp-Ser-Thr-Leu-Ala-Asp-Leu-Gly-Leu-Asp-Ser(4'-phosphopanteth ein e) -Leu-Met-Gly-Val-Glu-Val-Arg-COOH.     

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 5 from the dacA mutant strain of Escherichia coli (TMRL 1222)

The localization of the active site of penicillin-binding protein 5 from the dacA mutant of Escherichia coli strain TMRL 1222 has been determined. The protein was purified to homogeneity and labeled with [14C] penicillin G. The labeled protein was digested with trypsin, and the active site tryptic peptide was purified by a combination of gel filtration and high-pressure liquid chromatography. Sequencing of the purified [14C]penicilloyl peptide yielded the sequence Arg-Asp-Pro-Ala-Ser-Leu-Thr-Lys, which corresponds to residues 40-47 of the gene sequence (Broome-Smith, J., Edelman, A., and Spratt, B. G. (1983) in The Target of Penicillin (Hakenbeck, R., Holtje, J.-V., and Labischinski, H., eds) pp. 403-408, Walter de Gruyter, Berlin). The catalytic amino acid residue that forms a covalent bond with penicillin was identified by treating the purified [14C]penicilloyl peptide with a mixture of proteases and then separating the radioactive products using high-pressure liquid chromatography. Analysis of the radioactive peaks by amino acid analysis confirmed that it is the serine residue that reacts with the beta-lactam ring of penicillin.     

Histidine 235 of human sex hormone-binding globulin is the covalent site of attachment of the nucleophilic steroid derivative, 17 beta-bromoacetoxydihydrotestosterone

This article deals with the elucidation of the steroid-binding site of human sex hormone-binding globulin (SHBG). 17 beta-Bromoacetoxydihydrotesterone (BA-DHT) reacted with highly purified SHBG in a time-dependent and irreversible fashion. The interaction could be totally inhibited by the simultaneous addition of an excess of dihydrotesterone. At the completion of the reaction, the molar ratio of BA-DHT to SHBG was approximately unity. SHBG was affinity labeled with [14C]BA-DHT and submitted to acid hydrolysis. The released amino acids were evaluated on high performance liquid chromatography, and virtually all of the 14C was identified as 3-[14C]carboxymethylhistidine. Furthermore, [14C]BA-DHT-labeled SHBG was digested with trypsin, followed by isolation of the released tryptic peptides by reverse-phase high performance liquid chromatography. The 14C was localized to a single tryptic peptide. It contained 2' histidyl residues, corresponding to residues 235 and 251 in the known amino acid sequence of SHBG. Although most of the 3-[14C]carboxymethylhistidine, or its phenylthiohydantoin derivative, was trapped on the filter of the amino acid sequenator, sufficient radioactivity emerged to identify histidyl residue 235 as the labeled amino acid.     

Catalytic site of rat liver and bovine heart fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Identification of fructose 6-phosphate binding site

Fructose-6-P binding sites of rat liver and bovine heart Fru-6-P,2-kinase:Fru-2,6-bisphosphatase were investigated with an affinity labeling reagent, N-bromoacetylethanolamine phosphate. The rat liver enzyme was inactivated 97% by the reagent in 60 min, and the rate of inactivation followed pseudo-first order kinetics. The bovine heart enzyme was inactivated 90% within 60 min, but the inactivation rate followed pseudo-first order up to 80% inactivation and then became nonlinear. The presence of fructose-6-P retarded the extent of the inactivation to approximately 40% in 60 min. In order to determine the amino acid sequence of the fructose-6-P binding site, both enzymes were reacted with N-bromo[14C]acetylethanolamine-P and digested with trypsin; radiolabeled tryptic peptides were isolated and sequenced. A single 14C-labeled peptide was isolated from the rat liver enzyme, and the amino acid sequence of the peptide was determined as Lys-Gln-Cys-Ala-Leu-Ala-Leu-Lys. A major and two minor peptides were isolated from bovine heart enzyme whose amino acid sequences were Lys-Gln-Cys-Ala-Leu-Val-Ala-Leu-Lys, Arg-Ile-Glu-Cys-Tyr-Lys, and Ile-Glu-Cys-Tyr-Lys, respectively. In all cases, N-bromoacetylethanolamine-P had alkylated the cysteine residues. The amount of bromo[14C]acetylethanolamine-P incorporated into rat liver and beef heart was 1.3 mol/mol of subunit and 2.1 mol/mol of subunit, respectively, and the incorporations in the presence of Fru-6-P were reduced to 0.34 mol/mol of subunit and 0.9 mol/mol of subunit, respectively. Thus, the main fructose-6-P binding site of rat liver and bovine heart enzymes was identical except for a single amino acid substitution of valine for alanine in the latter enzyme. This peptide corresponded to residues 105 to 113 from the N terminus of the known amino acid sequence of rat liver enzyme, but since the complete sequence of bovine heart enzyme is not known, the location of the same peptide in the heart enzyme cannot be assigned.     

Clostridium perfringens iota toxin ADP-ribosylates skeletal muscle actin in Arg-177

Clostridium perfringens iota toxin ADP-ribosylates actin. Substrates of C. perfringens toxin are both non-muscle beta/gamma-actin and skeletal muscle actin. This finding suggests that C. perfringens iota ADP-ribosylates the same amino acid in skeletal muscle and non-muscle actin as does C. botulinum C2 toxin in non-muscle actin. Protein chemical analysis involving thermolysin cleavage on [32P]ADP-ribosylated actin or tryptic digestion followed by a secondary thermolysin cleavage of the radiolabelled fragments showed one major site of ADP-ribosylation. From its amino acid composition and sequence, the radiolabelled peptide was identified as peptide 175-177, locating the acceptor ADP-ribosyl amino acid as Arg-177.     

Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase

We reported previously that the ADP-ribosyltransferase in C1 and D botulinum toxins specifically catalyzes ADP-ribosylation of an Mr 22,000 guanine nucleotide-binding protein and that this substrate named Gb (b = botulinum) has an amino acid sequence homologous to that deduced from the rho gene (Narumiya, S., Sekine, A., and Fujiwara, M. (1988) J. Biol. Chem. 263, 17255-17257). In this study we have determined the amino acid sequence at its ADP-ribosylation site. Purified substrate was [32P]ADP-ribosylated by C1 botulinum toxin and digested with trypsin. The radioactive peptides were isolated by reversed-phase high performance liquid chromatography and digested further either with protease V8, with proteases V8 and thermolysin, or with proline endopeptidase and thermolysin. By this procedure three radioactive peptides were obtained, and their amino acid sequences were X-Tyr-Val-Ala-Asp-Ile-Glu, X-Tyr, and Val-Phe-Glu-X-Tyr in which no amino acid peak was found in X. During the sequencing the radioactivity quantitatively adhered to the sequencing filter and was not eluted with either of the identified amino acid residues. Analysis of the protein without the ADP-ribosylation yielded the corresponding sequence as Thr-Val-Phe-Glu-Asn-Tyr which corresponds to Thr37-Tyr42 in the amino acid sequence deduced from the Aplysia rho gene. These results strongly suggest that the asparagine residue is the ADP-ribosylation site in the rho gene product. This ADP-ribose protein bond was stable in 0.5 M hydroxylamine at pH 7.5 at 37 degrees C for at least 5 h. The ADP-ribosylation of this protein affected neither its GTPase- nor its [35S]guanosine 5'-O-thiotriphosphate-binding activity.     

Covalent modification of substrate-binding sites of Escherichia coli ADP-glucose synthetase. Isolation and structural characterization of 8-azido-ADP-glucose-incorporated peptides

A photoaffinity substrate analogue, 8-azido-ADP-[14C]glucose, reacts specifically and covalently with Escherichia coli ADP-glucose synthetase. The site(s) of reaction of 8-azido-ADP-[14C]glucose with the enzyme was identified by isolation of tryptic peptides containing the labeled analogue by use of high performance liquid chromatography technique and subsequent NH2-terminal sequence analysis of the purified radioactive peptides. One major binding region of the azido analogue is a peptide segment composed of residues 107-114 of the enzyme's polypeptide chain. Lys 108 and Arg 114 become trypsin-resistant sites when the enzyme is photoinactivated by 8-azido-ADP-[14C] glucose, suggesting that the analogue binds at or near the vicinity of these 2 basic amino acid residues. Conformational analysis of this peptide segment (residues 107-114) shows a strong probability of a reverse beta-turn secondary structure, suggesting that this peptide segment is on the enzyme surface. Two minor reaction regions of the enzyme with the analogue were also identified by chemical characterization. One region was composed of residues 162-207. Lys 194 was previously suggested as the activator-binding site by chemical modification studies with pyridoxal phosphate (Parsons, T. F., and Preiss, J. (1978) J. Biol. Chem. 253, 7638-7645). Another minor region where the analogue binds the tryptic peptide composed of residues 380-385 is near the COOH-terminal side of the enzyme. It is postulated that all these peptide segments are juxtaposed in tertiary structure.     

Amino acid sequence of the NAD (H)--binding region of the mitochondrial nicotinamide nucleotide transhydrogenase modified by N,N'-dicyclohexylcarbodiimide

Purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) is inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), and NAD(H) protects the enzyme against this inhibition [Phelps, D.C., and Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The tryptic digest of TH treated with [14C]DCCD showed a single radioactive peak upon FPLC chromatography. This radioactive peak was absent from tryptic digests of TH treated with [14C]DCCD in the presence of NADH. Sequence analysis of the radioactive peak showed that it contained two peptides, one derived from the other as a result of incomplete cleavage by trypsin of a lysyl-glutamyl bond. After further digestion with Staphylococcus V8 protease, the smaller radioactive fragment was isolated and sequenced. The amino acid sequence of this fragment, as determined by manual Edman degradation, was Ala-Glu-Met-Lys. The second residue was modified. Amino acid analysis and sequence studies on the radioactive tryptic peptide mixture indicated that the sequence around the DCCD-modified residue was Glu-Met-Ser-Lys-Glu-Phe-Ile-Glu-Ala-Glu-Met-Lys. In other studies, this sequence has been found in the amino acid sequence of TH as predicted from the corresponding cDNA. The DCCD-modified peptide is near the site of NAD(H) binding, as labeled with radioactive p-fluorosulfonylbenzoyl-5'-adenosine. Furthermore, there is a high degree of homology in this region between the amino acid sequences of the bovine heart TH and the alpha subunit of the Escherichia coli TH.     

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with [14C]penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the [14C]penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.     

Botulinum C2 toxin ADP-ribosylates cytoplasmic beta/gamma-actin in arginine 177

Isolated cytoplasmic actin of human platelet and pig liver actin, but not rabbit skeletal muscle actin, was ADP-ribosylated by botulinum C2 toxin in the presence of [32P]NAD. Tryptic digestion of the [32P]ADP-ribosylated platelet actin generated two labeled peptides: a soluble peptide covering residues 174-183 and an insoluble fragment containing residues 148-183. Further digestion of these two peptides with thermolysin yielded the same radioactive peptide, which was in both cases peptide 175-177. Amino acid sequence analysis of peptides 174-183 and 175-177 located the ADP-ribosylation on Arg177.